A fundamental question in biology is how biochemical processes in a cell are integrated to generate a balanced and robust metabolism. The long term goal of our research is to contribute to understanding metabolic integration by characterizing both new components and new connections in the metabolic network. The bacterium Salmonella enterica is used for these studies primarily because sophisticated genetic, biochemical and molecular techniques to be used to address complex metabolic questions. The extensive literature on biochemical pathways and metabolic properties of this organism (and that of its close relative E. coli) and the availability of an annotated genome sequence make it an optimal model system for metabolic studies. The thiamine biosynthetic pathway has proven to be a productive model system for these studies on metabolic integration. Work described in this proposal will further our understanding of the integration of thiamine biosynthesis with other metabolic processes. This will be done by i) defining the kinetic parameters of an enzyme that can contribute to both tryptophan and thiamine synthesis, ii) characterizing a locus encoding a new activity generating phosphoriobosyl amine, iii) defining the cellular components required for optimal conversion of aminoimidazole ribotide to the pyrimidine moiety of thiamine, and iii) defining the function of 4 orfs identified by our work. To accomplish the goals of this proposal, modern chemical, biochemical, molecular and genetic techniques will be used. The work herein will increase our understanding of the metabolic needs for thiamine synthesis under different conditions. This work will uncover new components in metabolism as the function of genes indirectly involved in thiamine synthesis are probed. This type of work is critical to the continued annotation of genomic sequence and understanding metabolism from a global perspective needed in genomic analysis. [unreadable] [unreadable]